The development of inhibitory antibodies against FVIII is not only a severe and important complication of protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Generation of such inhibitors might potentially preclude gene therapy for hemophilia A. In this project, we propose to investigate a novel gene therapy approach that will provide therapeutic FVIII protein and induce immune tolerance for hemophilia A and hemophilia A with inhibitors based on the hypothesis that targeting the production of FVIII to platelets that activate at the site where FVIII is needed could overcome the presence of inhibitory antibodies; in addition, platelets contain immunomodulatory molecule, transforming growth factor beta (TGF), would induce antigen-specific regulatory T cells, promoting immunologic tolerance. We have developed a clinically translatable gene therapy protocol for hemophilia A using lentiviral (LV) gene delivery of the FVIII expression cassette under control of the platelet-specific aIIb promoter (2bFVIII) to hematopoietic stem cells resulting in FVIII expression in platelets. Our studies have demonstrated that sustained therapeutic levels of platelet-FVIII expression are obtained while inhibitor titers decline with tie in 2bFVIIILV-transduced FVIII-primed hemophilia A mice (inhibitor model). Our most recent studies show that 2bFVIII gene therapy can not only restore hemostasis but also induce immune tolerance in the non-inhibitor model mice. In the current application, we propose to explore how immune tolerance is induced in hemophilia A mice after platelet gene therapy and the efficacy of this novel gene therapy approach in inducing immunologic tolerance in the clinical setting of pre-existing FVIII inhibitory antibodies. We will also investigate how the clinically relevant non-myeloablative conditioning regimens affect the immunologic consequences in platelet gene therapy of hemophilia A and hemophilia A with pre-existing anti-FVIII immunity. These studies should help us to further understand the biological characteristics of 2bFVIII gene therapy, with a potential developing a gene therapy approach that can not only provide therapeutic protein, but also induce the antigen-specific immune tolerance for the clinical treatment of HA patients and patients with inhibitors, as well as non-hereditary hemophilic patients with acquired inhibitory antibodies that can also have life-threatening clinica bleeding.